马铃薯Y病毒外壳蛋白基因的克隆及序列分析

本文报道应用聚合酶链式反应(PCR)技术,在体外扩增马铃薯 Y 病毒外壳蛋白基因及其克隆和序列分析的结果。病毒 RNA 从马铃薯 Y 病毒感染的烟草叶片中提取,用合成的PCR 3引物及 AMV 逆转录酶合成了单链的 cDNA。利用 PCR 技术,经30个循玎的扩增。得到了一特异的0.8kb 片段。克隆后对此片段进行了限制性内切酶物理图谱分析,并测定了其全序列。实验结果证明,我们克隆到的是完整的马铃薯 Y 病毒的外壳蛋白基因。与国外报道的马铃薯 Y 病毒 N 株相比,其核苷酸序列及推测的氨基酸序列的同源率分别为97.8%和97%。将该基因导入马铃薯以期获得抗 Y 病毒马铃薯的工作正在进行。本文还对 PCR 技术用于扩增植物 RNA 病毒的方法以及用基因工程方法培育抗病毒作物新品种的可行性等进行了讨论。     

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus.

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. These results indicate that the hydrophobic membrane-spanning region of gp52 is required for pathogenicity of SFFV and suggest that these sequences may play a role in signal transduction. The results also indicate that the transport defect of SFFV gp52 is due to structural features of the ectodomain of the molecule.     

Molecular analysis of yellow fever virus 17DD vaccine strain.

The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared worldwide. As part of a broader approach to determine the genetic variability in YF 17D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purified directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF 17D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot hybridization of virion RNAs of purified 17DD with two other strains of YF 17D virus revealed only genome-sized molecules for all three viruses. These observations suggest that the vaccine phenotype is primarily associated with the accumulation of mutations.     

On the effect of temperature and pH on the settling behaviour of a flocculent strain ofZymomonas mobilis

Summary In this paper the effect of temperature and pH on the settling behaviour of a flocculent strain ofZymomonas mobilis is studied by using the old fashioned batch settling technique. Plots are given to show the influence of the above mentioned parameters on the settling curve behaviour.     

gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.     

Biological control of Pythium damping‐off by seed‐treatment with pseudomonas putida: Relationship with ethanol production by pea and soybean seeds

The relationship between biocontrol activity of Pseudomonas putida strain N1R against Pythium ultimum on pea and soybean seeds and the reduction in ethanol evolution by imbibed seeds was investigated under different treatment conditions, including temperature and numbers of seed‐applied cells of the bacterium. Treatment with strain N1R increased emergence at all temperatures, except for soybean at 12 °C and reduced ethanol concentration in the spermosphere of imbibed seeds at several temperatures. The concentration of bacterial cells in the seed treatment suspension also significantly affected biocontrol efficiency and reduced ethanol production, especially in pea seeds. In contrast, the duration (0–7 h) of submergence of seeds in bacterial suspension had little effect on biocontrol activity of N1R, although submergence of soybean seeds reduced their emergence even in the absence of the pathogen or biocontrol agent. Competition for seed‐derived compounds, including ethanol, is suggested to be one possible mechanism of biocontrol of Pythium by strain N1R, which is not known to produce antifungal antibiotics.     

Glutamic acid decarboxylase of embryonic avian retina cells in culture: Regulation byγ-aminobutyric acid (GABA)

1. Retina-cell aggregate cultures expressed glutamate decarboxylase activity (L-glutamate 1-carboxylase; EC 4.1.1.15) as a function of culture differentiation. 2. Glutamic acid decarboxylase (GAD) activity was low in the initial phases of culture and increased eight-fold until culture day 7, remaining high up to day 13 (last stage studied). 3. The addition of GABA to the culture medium 24 h after cell seeding almost totally prevented the expression of GAD activity. 4. In association with decreased enzyme activity, aggregates exposed to GABA did not display immunoreactivity for GAD, suggesting that GAD molecules were either lost from GABAergic neurons or significantly altered with GABA treatment. 5. Control, untreated aggregates showed intense GAD immunoreactivity in neurons. Positive cell bodies were characterized by a thin rim of labeled cytoplasm with thickest labeling at the emergence of the main neurite. 6. Heavily labeled patches were also observed throughout the aggregates, possibly reflecting regions enriched in neurites. 7. The GABA-mediated reduction of GAD immunoreactivity was a reversible phenomenon and could be prevented by picrotoxin.